Influenza vaccine

#The present invention relates to a recombinant influenza neuraminidase, an expression vector with which the recombinant neuraminidase can be expressed in host cells, methods for producing and purifying recombinant neuraminidase, vaccines against influenza and the use of recombinant neuraminidase according to the invention.

#Influenza A and B virus epidemics cause considerable discomfort to those affected and have a great influence on social and economic life. They cause a significant mortality rate in older people and in patients with chronic illnesses. Since their introduction during the 1940's, inactivated vaccines based on virus material cultured in chicken eggs have been found to be clearly effective against influenza infection and have resulted in a significant fall in the mortality rate of high-risk populations.

#The influenza viruses are unique among the viruses of the bronchial tubes because they undergo a significant antigenic variation (so-called "drift") in their two surface antigens, that is, the hemagglutinin (HA) and the neuraminidase (NA).

#FIG. 1 shows the strategy for the construction of a secretable NA gene and its integration in a baculovirus transfer vector. Only the relevant restriction sites are indicated. The single lines show bacterial plasmide sequences, while the denser portions indicate HA-specific (full) or NA-specific (dotted) sequences. The HA signal sequence is indicated with single hatching. The NA signal sequence/membrane anchor sequence is indicated with double hatching.

#FIG. 2 shows the nucleotide sequence of the positive cDNA string and the amino acid sequence of the flanking regions of the ligation site between the HA signal peptide and the NA with its NA membrane anchor removed.

#FIG. 2A shows non-processed HA with, in detail, the signal peptidase restriction site between Ala.sub.16 and Gln.sub.1, (dotted vertical line). The NA terminal segment used for the secretion of NAs is designated with an arrow.

#TABLE 1 ______________________________________ Purification of pNA Vol- Total Specific Purifi- ume Protein activity Yield activity cation Steps ml mg U % U/mg fold ______________________________________ Crude pNA material 21.0 22.1 86,000 100 3,890 1.0 Sepharose S 32.5 0.98 53,800 62.6 54,900 14.1 Superdex 200 12.0 0.74 42,900 49.9 58,000 14.9 ______________________________________

#The table relates to a single typical purification experiment (see text for details). The volume after Superdex 200 gel filtration shows a pool of two chromatography runs.

#TABLE 2 ______________________________________ Purification of NAs produced by Sf9 insect cells Vol- Total Specific Purifi- ume Protein activity Yield activity cation Steps ml mg U % U/mg fold ______________________________________ Crude medium 995 281 144,000 100 510 1.0 (20-60)% 99.5 97.5 117,000 81.3 1,200 2.4 (NH.sub.4).sub.2 SO.sub.4 precipitate Sepharose Q 60.0 7.27 70,300 48.9 9,670 19.0 N-(p-aminophenyl) 54.0 2.63 49,100 34.1 18,700 36.7 oxamic acid agrose Superdex 200 tetramer 8.0 0.66 36,000 25.1 54,700 107 dimer 8.0 0.98 -- -- -- -- monomer 8.0 0.15 -- -- -- -- ______________________________________